Fluorescent multiparameter particle analysis

ABSTRACT

Methods are provided for rapidly determining a number of parameters in a few determinations. Particularly, the method is applicable to blood typing, determining the blood type as to the ABO and Rh type, as well as the determination of isoantibodies to the antigens. The method employs fluorescent particles having a plurality of fluorescers, where the presence or absence of light emission of a particular wavelength can be determined.

This is a continuation of application U.S. Ser. No. 482,124, filed onApr. 5, 1983, and U.S. Pat. No. 4,584,277.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The continued dependence upon whole blood obtained from individuals forreplenishing blood in another person requires the monitoring of largevolumes of blood for their blood group type. In determining the bloodgroup, one is interested in a number of factors: The particular type inthe ABO group; the presence of isoantibodies to the antigens of the ABOgroup; and the Rh type. Where each of these factors must be determinedindependently, a large number of tests are involved. For the most part,hemagglutination tests have been involved in measuring the variousfactors, which are subjective labor intensive and cumbersome.Furthermore, they have not readily lent themselves to automation, sothat the tests can be run rapidly with minimum involvement of atechnician. It is therefore desirable to find techniques which allow forminimal numbers of determination, automation of the method ofdetermination, while accurately reporting the information necessary forblood typing.

2. Brief Description of the Prior Art Hoffman et al., Int. J.Immunopharmac. (1981) 3(3): 249-254 describes immunofluorescent analysisof blood cells by flow cytometry. Methods for measuring fluorescentbeads may be found in Briggs et al., Science (1981) 212: 1266-1267 andNicoli et al., PNAS USA (1980) 77: 4904-4908. See also copendingapplication Ser. No. 397,285, filed July 12, 1982, which disclosuresincorporate herein by reference, as describing an alternate techniquefor measuring fluorescent cells. For a general discription, see FlowCytometry and Sorting, (eds. Melamed et al.) John Wiley and Sons, NewYork, 1979.

SUMMARY OF THE INVENTION

Methods and compositions are provided for assaying in bulk solution amultiparameter sample in a minimum number of determinations withoutemploying restricted flow or separations. The method employs particlesand fluorescent labels, where light signals are simultaneously orsequentially determined as indicative of a component in the assaymedium. Illustrative of the technique is the typing of a singlesuspension of a whole blood sample as to ABO group and isoantibodies,and the determination of the special A,B, isoantibodies and Rh factor.The former determination employs A and B phenotype particlesdistinguishable from native erythrocytes. Fluorescent antibodies areemployed where independently determined fluorescent measurements can berelated to the parameter of interest.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

In accordance with the subject invention, novel methods and compositionsare provided for determining a plurality of parameters in a sample witha minimum number of determinations. The method involves the use ofparticles and one or more fluorescers and the detection of at least twodistinguishable signals. Depending on the number of signals determinedbased on the same or different valued spectroscopic characteristics,with a single determination, two signals can distinguish two parameters.With an increasing number of signals, greater numbers of parameters maybe determined. In general, with n signals one can distinguish 2^(n-1)parameters. Usually not more than three signals will be employed,allowing for a determination of 4 parameters.

The determinations involve inspecting a single particle as to whether anobserved signal differs from a threshold value by a predetermined amountpositive or negative. Depending on the parameters of interest, therewill be populations of particles with signals above or below apredetermined threshold, which will define the presence or absence ofthe particular parameter. The parameters are epitopic sites and bindingsites of specific binding members which are ligands and receptors,respectively.

In many situations there is an interest in determining the presence,absence or coexistence of a plurality of parameters. For example, in HLAtyping, one can be interested in determining the presence or absence ofparticular combinations of subtypes. Another area of interest can be thedetermination of the presence of cell surface proteins. There is alsothe area of blood typing involving antigenic sites on cell surfaces andantibodies to phenotypes. Of course, this invention need not berestricted to cells, since there will be other situations where one isconcerned with the simultaneous presence of two parameters, which may bephysically associated or dissociated.

In performing the subject invention one uses a single suspension wheredye reagents, particularly fluorescent reagents, either the same ordifferent, may be added concomitantly or sequentially. One thendistinquishes between individual reagent molecules dispersed in solutionand groups of reagent molecules in relatively close proximity. Theassembling or grouping of the dye molecules on a particle provides for asubstantial difference in signal due to the substantially higherconcentration of dye reagent associated with an individual particlewhich is being monitored. By monitoring a sufficiently small volume, sothat on the average only one particle is being inspected at a time, andmaking a large number of measurements of such sized volume, whichmonitored volumes differ temporally or spatially, one determines theproportion of particles (i.e. volumes) associated with a signaldiffering from a predetermined threshold value. Based on the number ofvolumes providing signal(s) exceeding a predetermined value, one canidentify the presence or absence of a plurality of parameters.

The sample will be continuous, being still or stirred and not a movingstream. That is, one monitors a portion of a sample which remains in adiffusive relationship with the entire sample being measured.

The reagents which are employed will be dye, particularly fluorescerlabeled specific binding members and particles or unlabeled particles,so long as the particle reagent is distinguishable from particlespresent in the sample medium. The reagents may be the same as thebinding member having the parameter or analogous to such binding memberby having the same or substantially the same binding properties. Forexample, where the parameter is an antibody binding site, the reagentcould be on Fab fragment, F(ab')₂, etc. Where the parameter is thedeterminant site of a ligand, one could employ an oligopeptide havinganalogous binding properties, which can compete with the parameterepitopic site for a reciprocal receptor.

A wide variety of protocols may be employed depending upon the number ofparameters of interest and the choice of reagents. For example, for themeasurement X and anti-Y where X is bound to a particle, one candetermine X with only fluorescer labeled anti-X (F-anti-X) and anti-Ywith a Y-labeled particle and fluorescer labeled anti-Y (F-anti-Y),where the Y-labeled particle can be distinquished from the X-boundparticle.

The protocol would be adding F-anti-X to the sample and determiningwhether the F-anti X becomes bound to X-bound sample particles. One thenadds the Y-labeled particles and F-anti-Y and determines if the numberof fluorescent labeled particles has increased to a level indicative ofthe absence of anti Y. In this manner two sequential measurements aremade with the same sample and in the same apparatus. Employing acarousel, the additions and determinations can be automated.

There is the opportunity to use a single fluorescer, where sequentialadditions and sequential measurements are made. For example, considerthe measurement of a sample of particles which may or may not have Xand/or Y. One could first add F-anti-X and measure the number ofparticles which fluoresce above a predetermined threshold value. Onewould then add F-anti-Y (the two Fs are the same fluorescer) anddetermine the number of particles having a predetermined value greaterthan the value observed after the addition of F-anti-X. By havingsequential additions and measurements, the necessity for measuring lightat two different wavelengths can be obviated.

A particular application for the subject method is in blood typing wherevarious combinations of parameters are of interest. The following Table1 indicates various parameters of interest, the number of fluorophoresrequired and the nature of the reagents.

                  TABLE 1                                                         ______________________________________                                                    No. of                                                            Analytes    Fluorophores                                                                             Reagents                                               ______________________________________                                        A; B; αA; αB                                                                  2          M.sub.B.sup.A ; αA-F.sub.1 ;                                            αB-F.sub.2                                       A; αA 1          M-A; αA-F.sub.1                                  B; αB 1          M-B; αB-F.sub.1                                  A; B        2          αA-F.sub.1 ; αB-F.sub.2                    αA; αB (plasma)                                                               1          E-A; αA-F.sub.1 ; M-B; αB-F.sub.1          αA; αB                                                                        2          M.sub.B.sup.A ; α-F.sub.1 ;                                             αB-F.sub.2                                       αA; αB                                                                        2          M-A; M-B; αA-F.sub.1 ; αB-F.sub.2          A; Rh       2          αA-F.sub.1; αRh-F.sub.2                    A; B; Rh    3          αA-F ; αB-F.sub.2 ; αRh-F.sub.3      A; B; (A,B) 3          α(A,B)-F.sub.1; αA-F.sub.2;                                       αB-F.sub.3                                       (A,B); Rh   2          α(A,B)-F.sub.1 ; αRh-F.sub.2               A; B; αA; αB                                                                  2          M-A; M-B; αA-F.sub.1 ; αB-F.sub.2          ______________________________________                                    

All assays can be run with whole blood except where indicated. When onlyantigens are determined the assay may be run with isolated cells. Whenonly antibodies are determined the assay may be run with serum orplasma. M means particle distinct from an erythrocyte (E); e.g. ghost,vesicle, or latex that is detectiblely light or by means of anadditional fluorophore. αX means antiX antibodies. The combinationassays may provide for determination of 2 to 4 parameters in a singleassay medium, a single assay medium providing 4 parameters being themost efficient.

In blood typing one will normally be interested in obtaining the maximumamount of information from a single determination. In accordance withthe protocols of this invention one can determine a variety ofparameters of interest to blood typing. By having two independentdeterminations, one can determine the ABO type, the presence or absenceof isoantibodies to the A and B antigens, the Rh type and the special A,B type. (The special A, B type involves a small percentage of thepopulation where the A antigen binds only weakly to the usual antiserumfor detecting the A antigen.) The method requires the use of from two tothree labels which can be independently distinquished. Conveniently,these labels are fluorescers, which have emission characteristics whichare readily distinguishable. The method involves independently,conveniently simultaneously, determining the presence or absence of thedifferent labels on an individual particle and distinguishing nativeerythrocytes from A and B or AB phenotype particles.

The measurement is based on having three parameters, of which at leasttwo are fluorescers having different emission maxima, so that they canbe distinquished, while being capable of being excited by from 1 to 2light sources. A third parameter is involved which is associated with aparticle having the A and/or B antigens. The third parameter will afforda detectable distinction between the erythrocytes present in the bloodsample and the particle which serves as a reagent and has the A and/or Bantigens. This reagent will be referred to as M, and M refers to amarked particle, which marker or distinction from an erythrocyte may beinherent in the nature of the particle or may be as a result of afluorescer label bound to the particle. The distinction provides adetectable electromagnetic radiation signal different from the signalobtained with an erythrocyte.

One particulate reagent (M) can be A and B positive erythrocyte to whicha fluorescer is bound, either covalently or non-covalently e.g. throughan antibody, which reagent will be referred to as E-F₃, where thefluorescer bound to the erythrocyte will be referred to as F₃. Whenreverse typing for determining αA or αB, antibodies to the erythrocytewill be for A or B determinants, respectively.

Other particles may be used to which the A and B antigens may be boundand which permit discrimination between an erythrocyte particle and thesubject particle. Such particles include polymeric beads, such aspolysaccharides an addition polymers, liposomes and erythrocyte ghosts,where the particles may or may not be labeled. The labels may be variedwidely depending upon the nature of the particle and the distinguishingdetectable signal.

One signal is light scatter where the scatter observed with theerythrocyte is different from the scatter observed with the M. Anothersignal is fluorescence, where the fluorescer bound to the particle has adifferent emission maximum or polarization from the other fluorescerspresent in the assay medium. Alternatively, the endogenous fluorescenceor opacity of erythrocytes could provide the distinguishing parameter.

For light scatter one may use a number of different materials to labelparticles such as ghosts and liposomes. Colloidal metal or metalcompounds, colloidal carbon, inks, etc. may be used. Alternatively, onecan depend on the intrinsic light scattering difference betweenerythrocytes and erythrocyte ghosts, which scatter light lessefficiently.

In addition to the erythrocyte reagents, labeled antibodies will also beused where the antibodies are specific for the particular phenotype.

In the subject assays, individual particles will be detected and thespectroscopic characteristics of these particles determined. Bydetermining the presence or absence of the fluorescers on a particularparticle, one can determine the phenotype of the host erythrocytes, aswell as the presence of isoantibodies to the A and B antigens.

The following Table 2 is exemplary of the matrix of signals originatingfrom individual particles which is diagnostic of the ABO type, as wellas the presence of antibodies to the AB antigens, when employing αA-F₁,αA-F₂, and M(A)B.

                  TABLE 2*                                                        ______________________________________                                                  F.sub.1    F.sub.2                                                                             M                                                  ______________________________________                                        Blood Type                                                                    A           +            -     -                                              B           -            +     -                                              AB          +            +     -                                              O           -            -     -                                              Isoantibodies                                                                 αA    -            +     +                                              αB    +            -     +                                              αAαB                                                                          -            -     +                                              -           +            +     +                                              ______________________________________                                         *α intends antibody                                                     F.sub.1 bound to αA                                                     F.sub.2 bound to αB                                                     M bound to erythrocyte bearing A and B antigensM(A)B                          + and - mean an elevated or reduced signal in relation to a defined signa     level.                                                                   

In analysing Table 2, one should consider that there will be at leasttwo types of particles present in the sample: The marked particleshaving the A and B phenotypes and the host erythrocyte particle, whosephenotype is to be determined. The erythrocytes and marked particles canbe readily distinquished. To illustrate the situation using afluorescent marker F₃ on A and B particles, for A blood type, the hosterythrocytes will bind to αA-F₁. Therefore, when a host erythrocyte isobserved, the emission and excitation from such erythrocyte will be atthe wavelength band of F₁. The A and B phenotypic particle will beeither F₁ +,F₂ -,F₃ + or F₁ +,F₂ +,F₃ + depending on whether or not αBantibodies are present. A similar analysis will follow for the otherblood types.

The host erythrocytes will be distinguishable from the AB labeledparticles, which if an erythrocyte, will be labeled with F₃. Therefore,those erythrocytes which do not fluoresce at the excitation and emissionwavelength bands of F₃ will be the host erythrocytes and will bediagnostic for the presence of A and B antigens. When fluorescence isobserved from a particle where there is emission in the wavelength bandof F₃, one can determine whether there are antibodies to the A and/or Bantigens by an elevated or reduced signal in the excitation and/oremission wavelength band of F₁ and F₂. Where antibodies to both A and Bantigens are present, one would observe a reduced number of particleswhich fluoresce in the wavelength range of F₃ and also fluoresce in thewavelength ranges of F₁ and F₂.

As already indicated the A and B phenotype particle reagents will bedistinguishable from the naturally occurring erythrocyte by a propertywhich is detectable by a light signal. The light signal may be as aresult of fluorescence, the particle being labeled with a fluorescer, oras a result of light scatter, the particle scattering light differentlyfrom an erythrocyte.

In accordance with the subject method, one is able to detectcombinations of fluorescers which are present on single particles. Thiscan be as a result of sequential or simultaneous measurement of thelight emitted from a single particle. For simultaneous measurement, onewould employ a detection means which permits differentiation of thedifferent wavelengths resulting from the fluorescers and, as appropriatelight scatter from each individual particle. For other than simultaneousmeasurements, a statistical anaylsis would be employed determining theincidence of the presence of a particular fluorescer associated with anerythrocyte or the marked particle. In this measurement a physicalmarker is not required. Rather, the "marked" particle must be present insubstantially different (>10) concentration from the erythrocytes. Thismay be illustrated with a measurement for A and anti-A, where F-anti-Aand unlabeled anti-A erythrocytes are employed. Initially, one wouldcombine the blood sample and F-anti-A. One measures fluorescence where apositive result indicating the presence of A antigen is a predeterminedpopulation of particles having a fluorescent value above a thresholdvalue. The unlabeled A erythrocytes are then added at a concentration atleast equal to that of the host erythrocytes and a second fluorescentmeasurement is made. A negative result indicating the absence of anti-Awill be a predetermined population of particles in excess of thepopulation previously observed that have a fluorescent value above thethreshold value.

Additionally, as previously described, different antibodies, eachlabeled with the same fluorescer, can be added sequentially to asuspension of host erythrocytes and a statistical measurement of thenumber of fluorescent particles made after each addition. The advantage,as indicated, is a simpler optical system.

Of particular interest is the use of the technique and apparatusdescribed in copending application Ser. No. 397,285, filed July 12,1982. The invention relies on the use of optical fibers which canaddress volumes, which are sufficiently small so that only singleparticles are interrogated as to their fluorescence. By employingsplitters and appropriate filters, one can simultaneously measurefluorescent signals and light scatter at two or three differentwavelength ranges or of different polarization. Thus, one can determinethe concentration of particles which have a relatively large number ofeach of one or more of the different fluorescers. Since one is notconcerned with the concentration of an individual fluorescer on thesurface of the particle, but only whether a significant threshold numberof such fluorescent molecules are present, the system need onlydiscriminate between the different wavelength ranges and not as to theamount of fluorescence coming from the particle.

A useful device is exemplified in copending application Ser. No.397,285, which may be employed without modification for sequentialmeasurements. For simultaneous measurements, the device may be modifiedin accordance with the following description. The device has a sampleholding means in which is immersed one or more optical fibers, which aredivided into one to three brancehs, each branch having separate filtersand/or polarizers which allow for the transmission of a signalcorresponding to the fluorescence emission or light scatter of one ofthe fluorescers. Excitation light may be introduced through one of thebranches or independently through a second optical fiber whichilluminates the sample at the optical fiber probe face. Where the lightis transmitted through the probe, a further fiber branch will beemployed for providing the light source. Another less preferred way isto employ two sources that could be used to excite at differentwavelengths. One could then employ branches for introducing excitationlight and eliminate the branches for the emission light.

A variety of sources of excitation light may be employed, preferablylasers, more particularly He-Cd, He-Ne or Ar lasers. Broad band lightsources must be very intense and filters must be employed to ensure theproper wavelength range to avoid enhancing background interference. Thelight source should be small and the light beam directed to the areadirectly in front of the optical fiber probe.

The emission light which is received by the probe and transmittedthrough the branches will be received by a detector. The detector is anydevice capable of receiving photons and converting them to a signal formwhich permits differentiation between signals of different intensities.A photomultiplier is a typical example.

The electrons emitted in one photo-pulse by a photomultiplier tube maybe directed to a preamplifier discriminator which amplifies the signal,discriminates against noise originating in the photomultiplier tube andgenerates a well-formed voltage pulse which may be counted by a digitalcounter. The number of photo-pulses per counter gate time isproportional to the intensity of light averaged over the gate time.These photo-pulse count values are interfaced to a computer which isprogrammed to detect changes in the count values, signifying a sharpfluctuation of fluorescence corresponding to the passage of a particleof interest through the effective sample volume. This is one example ofhow the signal from the light detector may be digitally analyzed.Alternatively, one could derive an analog signal from the detector anddetect sharp transitions with a high-pass filter, or, combinations ofanalog and digital techniques can be used.

The frequency of fluctuations in the signal exceeding a threshold valueis calculated and related to known calibrators. One can then determinethe percentage of particles observed which have the various combinationsof fluorescence emission for example as described above in Tables 1 and2. The computer can then be programmed to automatically report the bloodtype in accordance with the observed fluorescent combinations above apredetermined threshold value.

In performing the subject invention, one or a plurality, usually notmore than three, fluorescers are required. The fluorescer which may beconjugated to the erythrocyte is the least critical of the fluorescersfor the following reasons. First, the fluorescer (F₃) can be conjugatedin relatively large amounts to the erythrocyte. Therefore, where suchfluorescer has a relatively low fluorescence efficiency, a greateramount of the fluorescer may be employed. Secondly, one only needs asufficient amount bound to the erythrocyte, which will allow forassurance of its presence in combination with the other fluorescers.Therefore, one may choose a wide variety of fluorescers, which areprimarily limited by not interfering with detection of the other twofluorescers. Conveniently, F₃ may have emission characteristics, wherethe emission maximum is less than 700, preferably less than 600, andmore preferably between 450 and 510 nm.

The other fluorescers should have noninterfering emission maxima,generally having maxima different by at least about 20 nm, preferably byat least about 25 nm and should have high fluorescence efficiencies, becapable of binding to proteins without being detrimentally affected bythe binding, as well as being minimally affected by non-specificinterference. The fluorescers should have emission maxima greater than450, preferably greater than 475, and more preferably greater than 500,where desirably the emission maxima of one will be in the range of about500-575 and the emission maxima of the other will be in the range ofabout 550-625 nm. Fluorescers of particular interest may be found in EPOApplication Ser. Nos. 80106587.1 and 80105253.1. Other fluorescers ofinterest include Texas Red, phycobiliproteins, derivatives of rhodamine,e.g., X-RITC, etc. The manner of conjugation of the fluorescers to theerythrocytes or the antibodies is widely described in the literature andneed not be exemplified here. See for example, U.S. Pat. Nos. 4,199,559and 4,318,846.

As an illustration of the use of three dyes, the first dye could befluorescein or succinyl fluorescein; the second dye,2,7-dimethoxy-4,5-dichloro-3',6'-dichloro-4' or 5'-carboxyfluorescein;and the third dye, Texas red. These dyes could be excited at 442 nm(He-Cd laser). Emission measurements would be made at 510±5, 560±15 and610±15 nm respectively.

Alternatively, one may use coupled dyes, where a first dye or sensitizerabsorbs light at shorter wavelengths and is capable of transferringenergy to a second dye which is capable of fluorescence. In this way,one can use combinations of dyes, where one dye has high efficiency ofabsorption and will excite another dye which absorbs at higherwavelengths and has a high fluorescence efficiency. Sensitizers whichfind use include compounds of the following structure. ##STR1## Thesensitizers may be used by direct bonding to a fluorescer or by bondingto the molecule to which the fluorescer is bonded.

In preparing the subject conjugates, the following is an exemplaryprocedure.

To a cooled solution (0°-5°) of anti-A (7.5 mg) in 0.5 ml of 0.05M PO₄³⁻ buffer pH 8.0 is slowly added a solution of N-hydroxy succinimide(NHS) ester of fluorescein (0.07 mg) in 25 μl DMF during 20 minutes.Stirring is continued overnight in the cold room. Next day the solutionis centrifuged for 2 minutes and the yellow solution is purified over aSephadex G-25 column using 0.05M PO₄ ³⁻ buffer pH 8.0. The faster movingconjugate (1.5 ml) is easily separated. The conjugate has a λ_(max)^(abs) 490 nm.

A sensitizer like the merocyanine compound can be attached through a--CO₂ H acid derivative, such as shown below: ##STR2## The abovecompound will be activated as the NHS ester and the NHS ester will beused to label antibodies as described above for labeling dyes.

In carrying out the blood typing, one may combine the reagentcompositions with the blood sample sequentially or simultaneously. Theblood sample may be used whole, but will normally be diluted by a factorof up to 10³, usually up to about 10², in the assay medium. The sampleand the reagents will normally be mixed in an aqueous buffered solution,generally at a pH in the range of about 5.0 to 9.5, which may include avariety of other materials, such as stabilizers, salts, inert powders,proteins, etc. The mixture will then be incubated for a sufficient timeto allow for binding of the various antibodies to the determinant sites.Usually, at least thirty seconds will be employed and not more thanabout one hour, generally thirty minutes will suffice. One need not havereached equilibrium, since the primary concern is that there besufficient binding to available antigenic determinants to allow for asufficiently strong signal for a positive determination. The sample isthen excited with appropriate light and the fluorescence emissiondetermined, and light scatter or absorption determined, as appropriate.By analyzing for the combinations of fluorescence, in combination withthe marker distinguishing the particle, one can determine the blood typeand the presence of antibodies to the antigenic determinants for the ABOsystem. Or, if desired, other combinations of analytes.

In accordance with the subject invention, a rapid efficient method isprovided for multiparameter analysis, such as in a single sample bloodtyping. The method allows for automation, so that determinations can becarried out quickly, efficiently, and with a minimum of technicianhandling. Results can be automatically computed and printed, so that thesample and results are easily and accurately related.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claims.

What is claimed is:
 1. A method for determining a plurality of bloodtyping parameters, said parameters being epitopic sites and bindingsites of specific binding members (SBM₁, SBM₂, . . . SBM_(m)), saidbinding members being ligands and reciprocal receptors respectively,wherein at least one parameter containing specific binding member or itsreciprocal specific binding member is bound to a particle (SBM-P orRSBM-P),said method involving at least one fluorescent label bound to aspecific bindng member (SBM-F) and at least one particle (P), differentfluorescent labels and different particles being distinguishable byspectroscopic characteristics, which are emission, absorption and lightscattering; said method comprising: combining in a liquid medium, (a) ablood sample having at least two blood typing parameters to be measured;(b) for parameter containing specific bindng members bound to a particle(SBM₁ -P₁, SBM₂ -P₂, . . . SBM_(m) -P_(m)), fluorescent labeled specificbinding member reciprocal to said specific binding member bound to saidparticle (RSBM₁ -F₁, RSBM₂ -F₂, . . . RSBM_(m) -F_(m)); (c) forparameter containing specific binding members not bound to a particle(SBM₁, SBM₂, . . . SBM_(m)), (1) a particle bound to a specific bindingmember reciprocal to said parameter containing specific binding member(RSBM₁ -P₁, RSBM₂ -P₂, . . . RSBM_(m) -P_(m)) and (2) a fluorescentlabeled specific binding member analogous to said parameter containingspecific binding member (SBM₁ -F₁, SBM₁ -F₁ . . . SBM_(m) -F_(m));wherein the number of different particles (P_(m)) and fluorescent labels(F_(m)) are chosen to give signals (n) that distinguish each parameterin said sample and wherein sequential additions and sequentialmeasurements may be made or simultaneous additions and simultaneousmeasurements may be made, and wherein n signals can distinguish 2^(n-1)parameters and wherein reciprocal binding members bind with each other;irradiating at least a portion of said medium with light, wherein saidmedium is continuous and said particles are suspended in said continuousmedium, and determining populations of particles having electromagneticsignals differing from threshold values; and relating said populationsto the presence of said parameters in said sample.
 2. A method accordingto claim 1, wherein said sample is whole blood.
 3. A method according toclaim 2, wherein said parameters are determinant sites on anerythrocyte.
 4. A method according to claim 1, having two particleswhich differ by distinguishable electromagnetic signals produced uponirradiation with light.
 5. A method according to claim 4, where one ofsaid particles is labeled with a fluorescer and the other of saidparticles is not labeled with a fluorescer.
 6. A method according toclaim 5, wherein two particles are present, one particle is anerythrocyte from said sample and the other particle is a fluorescerlabeled erythrocyte.
 7. A method according to claim 1, whereinindividual particles are monitored for their fluorescence within apredetermined wavelength range by monitoring a sample volumesufficiently small to have a high probability of having no or oneparticle.
 8. A method for determining a plurality of blood typingparameters, said parameters being epitopic sites and binding sites ofspecific binding members (SBM₁, SBM₂, . . . SBM_(m)), said bindingmembers being ligands and reciprocal receptors, respectively, wherein atleast one parameter containing specific binding member is bound to aparticle (SBM-P),said method involving one fluorescent label bound to atleast one member (SBM-F) and at least one particle (P); said methodcomprising: combining in a liquid medium, (a) a blood sample having atleast two blood typing parameters to be measured; (b) in sequentialadditions as to each parameter:(i) where a parameter containing specificbinding member is bound to a particle (SBM₁ -P₁, SBM₂ -P₂, . . . SBM_(m)-P_(m)), fluorescer labled specific binding member reciprocal to saidparameter contaning specific binding member (RSBM₁ -F₁, RSBM₂ -F₂, . . .RSBM_(m) -F_(m)); (ii) where a parameter containing specific bindingmember is unbound (SBM₁, SBM₂, . . . SBM_(m)), (1) a particle having thereciprocal specific binding member (RSBM₁ -P₁, RSBM₂ -P₂, . . . RSBM_(m)-P_(m)); and (2) a fluorescer labeled specific binding member analogousto said parameter containing specific binding member (SBM₁ -F₁, SBM₂-F₂, . . . SBM_(m) -F_(m)); wherein the number of different particlesand fluorescent labels are chosen to give signals (n) that distinguisheach parameter in said sample and wherein sequential additions andsequential measurements may be made or simultaneous additions andsimultaneous measurements may be made, and wherein n signals candistinguish 2^(n-1) parameters and wherein reciprocal binding membersbind with each other; with the proviso that, when only the fluorescerlabeled specific binding member (SBM-F) is added in the second addition,there will be at least about a 2-fold increase in binding eventsrelative to the binding events that have already occurred; and when aparticle (P) is added in the second addition the number of particles inthe medium will be at least about doubled; irradiating at least aportion of said medium with light, wherein said medium is continuous andsaid particles are suspended in said continuous medium; determining thepopulation of particles differing from a threshold value as to anelectromagnetic signal after the first addition and the population ofparticles differing from a threshold value as to an electromagneticsignal after the second addition and so on for each parameter in saidsample; and relating said populations to the presence of said parametersin said sample.
 9. A method for determining a plurality of parameters,said parameters being epitopic sites and binding sites of specificbinding members (SBM₁, SBM₂, . . . SBM_(m)), said binding members beingligands and reciprocal receptors respectively, wherein at least oneparameter containing specific binding member or its reciprocal specificbinding member is bound to a particle which is an erythrocyte (SBM-P orRSBM-P),said method involving at least one fluorescent label bound to aspecific binding member (SBM-F) and at least one particle (P), differentfluorescent labels and different particles being distinguishable byspectroscopic characteristics, which are emission, absorption and lightscattering; said method comprising: combining in a liquid medium, (a) asample having at least two parameters to be measured; (b) for parametercontaining specific binding members bound to a particle (SBM₁ -P₁, SBM₂-P₂, . . . SBM_(m) -P_(m)), fluorescent labeled specific binding memberreciprocal to said specific bindng member bound to said particle (RSBM₁-F₁, RSBM₂ -F₂, . . . RSBM_(m) -F_(m)); (c) for parameter containingspecific binding members not bound to a particle (SBM₁, SBM₂, . . .SBM_(m)), (1) a particle bound to a specific binding member reciprocalto said parameter containing specific binding member (RSBM₁ -P₁, RSBM₂-P₂, . . . RSBM_(m) -P_(m) and (2) a fluorescent labeled specificbinding member analogous to said parameter containing specific bindingmember (SBM₁ -F₁, SBM₂ -F₂ . . . SBM_(m) -F_(m) ; wherein the number ofdifferent particles (P_(m)) and fluorescent labels (F_(m)) are chosen togive signals (n) that distinguish each parameter in said sample andwherein sequential additions and sequential measurements may be made or,simultaneous additions and simultaneous measurements may be made, andwherein n signals can distinguish 2^(n-1) parameters and whereinreciprocal binding members bind with each other and wherein individualparticles are monitored in a non-flow system for their fluorescencewithin a predetermined wavelength range by monitoring a sample volumesufficiently small to have a high probability of having no or oneparticle; irradiating at least a portion of said medium with light,wherein said medium is continuous and said particles are suspended insaid continuous medium, and determining populations of particles havingelectromagnetic signals differing from threshold values; and relatingsaid populations to the presence of said parameters in said sample. 10.A method according to claim 9, wherein said sample is whole blood.
 11. Amethod according to claim 9, having two particles which differ bydistinguishable electromagnetic signals produced upon irradiation withlight.
 12. A method according to claim 11, where one of said particlesis labeled with a fluorescer and the other of said particles is notlabeled with a fluorescer.
 13. A method according to claim 12, whereintwo particles are present, one particle is an erythrocyte from saidsample and the other particle is a fluorescer labeled erythrocyte.
 14. Amethod for determining a plurality of parameters, said parameters beingepitopic sites and binding sites of specific binding members (SBM₁,SBM₂, . . . SBM_(m)), said binding members being ligands and reciprocalreceptors, respectively, wherein at least one parameter containingspecific binding member is bound to a particle (SBM-P),said methodinvolving one fluorescent label bound to at least one member (SBM-F) andat least one particle (P); said method comprising: combining in a liquidmedium, (a) a sample having at least two parameters to be measured; (b)in sequential additions as to each parameter:(i) where a parametercontaining specific binding member is bound to a particle (SBM₁ -P₁,SBM₂ -P₂, . . . SBM_(m) -P_(m)), fluorescer labeled specific bindingmember reciprocal to said parameter containing specific binding member(RSBM₁ -F₁, RSBM₂ -F₂, . . . RSBM_(m) -F_(m)); (ii) where a parameetercontaining specific binding member is unbound (SBM₁, SBM₂, . . .SBM_(m)), (1) a particle having the reciprocal specific binding member(RSBM₁ -P₁, RSBM₂ -P₂, . . . RSBM_(m) -P_(m)); and (2) a fluorescerlabeled specifc binding member analogous to said parameter containingspecific binding member (SBM₁ -F₁, SBM₂ -F₂, . . . SBM_(m) -F_(m));wherein the number of different particles and fluorescent labels arechosen to give signals (n) that distinguish each parameter in saidsample and wherein sequential additions and sequential measurements maybe made or simultaneous additions and simultaneous measurements may bemade, and wherein n signals can distinguish 2^(n-1) parameters andwherein reciprocal binding members bind with each other and whereinindividual particles are monitored in a non-flow system for theirfluorescence within a predetermined wavelength range by monitoring asample volume sufficiently small to have a high probability of having noor one particle; with the proviso that, when only the fluorescer labeledspecific binding member (SBM-F) is added in the second addition, therewill be at least about a 2-fold increase in binding events relative tothe binding events that have already occurred; and when a particle (P)is added in the second addition the number of particles in the mediumwill be at least about doubled; irradiating at least a portion of saidmedium with light, wherein said medium is continuous and said particlesare suspended in said continuous medium; determining the population ofparticles differing from a threshold value as to an electromagneticsignal after the first addition and the population of particlesdiffering from a threshold value as to an electromagnetic signal afterthe second addition and so on for each parameter in said sample; andrelating said populations to the presence of said parameters in saidsample.